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Annular beam shaping - a theoretical investigation of the effect on the point-spread function in two-photon microscopy

Poster (konferens)
Författare Johan Borglin
Publicerad i Lasers in Medicine, 22-27/7 2012 Plymouth, NH, USA
Publiceringsår 2012
Publicerad vid Institutionen för fysik (GU)
Språk en
Ämnesord Two photon microscopy, simulation
Ämneskategorier Medicinsk bildbehandling


Two-photon fluorescence microscopy (TPM) is a well-established method for high resolution, non-invasive investigations of biological tissue. Working in the near infrared spectral region, the absorption in tissue is kept at a minimum. The non-linearity and low probability of the two-photon excitation process ensures that the detected signal is only generated within the focal volume where the photon flux is high. Recent applications of the technique demonstrate the feasibility for annular beam shaping in combination with TPM, e.g. for laser sheet microscopy. Annular beams will create Bessel beams which will carry special characteristics, e.g. elongated focal volume. To correctly implement annular beam shaping in TPM it is necessary to have full control on the shape and size of the focal volume, i.e. the point spread function (PSF). In this theoretical work, we investigate how the shape and dimensions of annular laser beams affect the generated PSF in TPM. The simulations are performed by implementing the Fresnel–Kirchhoff diffraction integral in MATLAB. We investigated how the ratio between the inner and the outer diameter of the beam affects the PSF of the beam in the focal plane. The results demonstrate that the focal area resembles that of an ordinary Gaussian beam for an inner radius of up to 40-50% of the full radius, when using an objective lens in the range of 0.8 to 1. When the inner radius of the beam exceeds this value, the focal volume becomes elongated in the axial direction, approaching a Bessel beam. This result implies that annular beam-shaping using a small inner radius can be applied for conventional TPM, while a larger inner radius of the annulus should be applied when elongated focal volume is desired, as in laser sheet microscopy.

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